The invention relates to the deprotonation of multiply charged fragment ions obtained by electron transfer dissociation of multiply charged analyte ions.
A common method of generating molecular ions for mass-spectrometric analysis of biomolecules is electrospray ionization (ESI), which ionizes molecules under atmospheric pressure outside the mass spectrometer. These ions are then channeled through inlet systems of a known type into the mass spectrometer's vacuum system, and on to the mass analyzer. There the ions are separated by mass and measured, yielding a mass spectrum of the biomolecule ions.
Electrospray ionization causes almost no fragmentation, and the ions are substantially those of the protonated molecules; due to their protonation, they are frequently referred to as “pseudomolecular ions”. Nevertheless, multiple protonation from electrospraying usually results in multiply charged ions of the molecules: doubly and triply charged ions for smaller molecules such as peptides, while for larger biomolecules, such as proteins, with molecular masses in the range between 5 and 20 kilodaltons, the ions may carry up to 10 or even 30 charges.
The absence of almost any fragmentation in the ionization process limits the information from the mass spectrum to the mass of the molecule. In most cases the enormous variety of biomolecules means that this is inadequate for the purposes of identifying the substance. The mass spectra do not contain further information about internal molecular structures which could be used to identify the substance under investigation. This information can only be obtained in special tandem mass spectrometers by recording the mass spectra of the fragment ions, which are obtained by fragmenting the molecular ions. A variety of methods are available for the fragmentation, and these depend strongly on the type of mass spectrometer being used.
If possible, fragmentation is carried out on parent ions with double or triple charges, as these have a very high yield of fragment ions and deliver easily evaluated fragment ion spectra. The spectra of these fragment ions are also known as “daughter ion spectra” of the parent ions concerned. It is also possible to measure “granddaughter ion spectra”, which are the fragment ion spectra of selected daughter ions. The structures of the fragmented ions can be read from these daughter (and granddaughter) ion spectra; for instance, it is possible (although somewhat difficult) to determine at least parts of the sequence of amino acids in a peptide from these spectra.
Mass spectrometers with RF ion traps have features that make them interesting for many types of analysis. In particular, they can isolate selected types of ion (the “parent ions”) in the ion trap and fragment them. The isolation of one type of ion means that all the uninteresting ion types are removed from the ion trap by strong resonant excitation or other measures, so that only the parent ions remain. These parent ions, in other words the interesting analyte ions, are fragmented following the conventional method, by weak resonant excitation of what are known as the “secular” ion oscillations, using a dipolar alternating voltage, which results in many impacts with the collision gas, but without removing the ions from the ion trap. The ions can accumulate energy through these impacts, finally resulting in decomposition of the ions and the creation of fragment (or daughter) ions. Until a few years ago, this collision-induced dissociation (CID) was the only known method of fragmentation in ion traps.
Three-dimensional (3D) Paul ion traps consist of a ring electrode and two end cap electrodes. As a general rule, the RF voltage is applied to the ring electrode, but other operating modes are possible. Ions of both polarities, i.e. positive or negative ions, can be held in the quadrupole RF field inside the ion trap for analysis by mass spectrometry. The ion traps can be used as mass spectrometers by ejecting the stored ions—selected according to mass—and measuring them in a so called “ion scan” with secondary electron multipliers. Several different ion scan methods are known for the ion ejection, but these will not be considered in any further detail here.
Linear ion traps (also known as 2D ion traps because the electrical fields in the interior only change in two dimensions) consist of several pairs of pole rods supplied with RF voltage, and end electrodes whose potentials can repel the ions. Special steps must be taken if it is desired to store both positive and negative ions at the same time; RF voltages, for instance, can be used to generate pseudopotentials that repel ions of both polarities. Two-dimensional ion traps with four pole rods form an internal quadrupole field, and can be used as mass analyzers in a similar way as 3D ion traps. Here again there are different scanning procedures, such as those using the mass-selective ejection of ions through slots in the pole rods, or through diaphragms at the end of the rod system.
Recently, a method has become known for the fragmentation of ions in ion traps that delivers the same kind of fragmentation as the now well-known electron capture dissociation (ECD) but by means of different reactions: electron transfer dissociation (ETD). This fragmentation process can be performed in ion traps by introducing suitable negative ions in addition to the stored analyte ions. Methods of this type have been described in the published patent applications DE 10 2005 004 324.0 (R. Hartmer and A. Brekenfeld) and US 2005/0199804 A1 (D. F. Hunt et al.). The fragment ions here (as in the case of electron capture) belong to what are known as the c and z series, and are therefore very different from the fragment ions of the b and y series, which are obtained by collision-induced fragmentation. The fragments in the c and z series have significant advantages for the identification of proteins and for determining the amino acid sequence from the mass-spectrometric data, not least because ETD fragment ion spectra can extend down to smaller masses than collision-induced fragment ion spectra.
It is most favorable if both collision-induced fragment ion spectra and ETD fragment ion spectra are recorded, as comparison of the two spectra permits the ion signals to be assigned immediately to the c/b series or to the z/y series. This is because there are fixed mass differences between c-ions and b-ions, as there are between z-ions and y-ions, which enable easy identification.
This fragmentation by electron transfer in reactions between multiply charged analyte cations and suitable anions is a favorable alternative to electron capture fragmentation (ECD), which is very difficult to carry out in ion traps because the RF fields scarcely permit the entry of low-energy electrons. RF ion traps can, however, store both positive and negative ions for the necessary electron transfer reactions in their pseudopotential wells.
The presence of a collision gas in the ion trap has the effect of damping the existing oscillations (the “secular” oscillations) of the ions in the pseudopotential well; the ions then collect after a few milliseconds as a small cloud in the center of the ion trap. In the case of a usual ion trap with typical ion content of a few tens of thousands of ions, the cloud has a diameter of about one millimeter; it is determined by equilibrium between the restoring force of the pseudopotential and the repulsive Coulomb forces between the ions. The internal dimensions of the 3D ion traps are generally characterized by a distance of about 14 millimeters between the end caps, the diameter of the ring electrode being somewhere between 14 and 20 millimeters. In linear quadrupole ion traps, the distance between opposing pole rods is generally around 8 millimeters; greater distances can, however, yield good 2D ion traps, particularly for linear hexapole or octopole ion traps.
The fragmentation of ions by electron transfer in an RF ion trap is created in a very simple manner by reactions between multiply charged positive ions and suitable negative ions. Suitable negative ions are often radical anions, such as those of fluoranthene, fluorenone, anthracene or other polyaromatic compounds. In radical anions, the chemical valences are not saturated, so facilitating the easy donation of electrons. They are generated in NCI (negative chemical ionization) ion sources, most probably through simple electron capture or through electron transfer. In principle, the design of NCI ion sources is the same as for chemical ionization (CI ion sources), but they are operated in a different way in order to obtain large quantities of low-energy electrons. NCI ion sources are also referred to as electron attachment ion sources.
The electron transfer reactions either result immediately in the desired fragmentation or, in an essentially undesirable manner, in the formation of radical cations of the analyte molecules. Although these radical cations have acquired an electron, they have not lost protons, and have therefore also not decomposed. These radical cations are inherently metastable, and therefore decompose over a sufficiently long period, thereby remaining intact in the ion trap for a relatively long time. They can very easily be subjected to further collision-induced fragmentation through gentle resonant excitation of their secular oscillations. This creates the fragment ions characteristic of electron transfer dissociation (ETD), and not the fragment ions typical of (CID) collision-induced fragmentation
The ETD fragment ion spectra are very easy to evaluate if they are produced from doubly charged parent ions. The evaluation of ETD fragment ion spectra from triply charged parent ions is also relatively simple, as doubly charged fragment ions are relatively easy to recognize by the differences in mass of their isotope patterns. This is not the case when highly charged parent ions having, for instance, ten or twenty charges, are subjected to this fragmentation procedure. The yield of fragment ions is then very high, but the fragment ion spectrum is so complex that it is scarcely possible to evaluate it, particularly as the isotope patterns in ion traps can no longer be resolved by mass, and therefore the level of charge cannot be established.
Larger molecules, proteins in particular, yield multiply charged ions in electrospray ion sources; as a rule of thumb, we can assume that every increase of 1500 Daltons in mass results in an average increase in charge of one elementary charge unit. A protein with a mass of 10000 Daltons has therefore gathered about 15 protons at the peak of the charge distribution, although in most cases there is a broad distribution of ions with various numbers of charges. Doubly or triply charged ions occur with vanishingly small frequencies, and therefore cannot practicably be used for generating the fragment ions; for these reasons, the use of fragmentation by electron transfer comes up against great difficulties with protein molecules in the molecular mass range between five and 50 kilodaltons, even though the highly charged analyte ions can be dissociated by electron transfer very effectively. In most cases, the fragment ions created in this way, above all the heavy fragment ions, are themselves also highly charged.
It has long been known that ions with multiple charges can be converted by continued deprotonation (“charge stripping”) into ions with single or low numbers of charges. This is done very easily by proton transfer from the ions with multiple positive charges to special kinds of negatively charged ions, most particularly non-radical anions, which are thereby neutralized. The reaction cross-sections for these proton transfer reactions are proportional to the square of the number of proton charges on an ion; the deprotonation therefore happens very quickly for highly charged ions, while the reaction speed is sharply reduced when the ions have lower charges. If, for instance, the supply of negative reactant ions for deprotonation is stopped when singly charged ions are reached, the measurements in the mass analyzer will demonstrate relatively simple mass spectra, as these now contain almost exclusively the signals of singly charged ions. Stopping the deprotonation reactions at an earlier stage, however, for instance when only mixtures of fragment ions with up to four protons remain, also leads to interpretable mass spectra if isotope resolution for scanning is achieved in the mass spectrometer used.
This effect can also be used when electron transfer dissociation is applied to proteins: after storing highly charged ions of the proton molecules, collision-induced fragmentation is produced by resonant excitation of the secular oscillations, or ETD fragmentation is generated by supplying suitable radical anions; after this, non-radical anions are supplied for deprotonation until the desired reduction in the charge states of the fragment ions has occurred. This yields easily interpretable fragment ion mass spectra.
If this method is applied to the deprotonation of ETD fragment ions it is unfavorable that, in principle, three different ion sources are required: one ion source for the analyte cations with multiple positive charges (usually electrospray), one ion source for generating the radical anions for the ETD reactions, and one ion source for the non-radical anions for deprotonation. If only one ion source is used to generate both kinds of anions, it is necessary, under the constraints of the technology known so far, to supply it with two different kinds of substance. The two substances can be supplied one after the other, but this is necessarily time-consuming and inconsistent with a rapid sequence of measurements. It can, alternatively, be done simultaneously, but this requires an additional selection of the desired ion types requiring additional procedures and equipment.